Determinants of promoter-specific activity by glucocorticoid receptor.

Glucocorticoid receptor (GR) is expressed at essentially equal levels in almost all tissues and cell types. Remarkably, glucocorticoids themselves regulate transcription in vivo in both a promoter- and tissue-specific manner. Thus, specific systems must be in place to regulate receptor action within certain cells and at certain promoters. To address two specific aspects of these systems, we have analyzed promoter-specific activity of GR using two different, well studied promoters (termed simple and composite promoters) from which GR activates transcription. The simple promoter depends only on the receptor for glucocorticoid-responsive transcriptional activation, while GR activity at the composite promoter depends on additional transcription factors. We have compared the action of several GR ligands at these promoters and demonstrate fundamental differences in the activities of these ligands on receptor activity. Furthermore, these compounds induce unique conformational changes in receptor, resulting in promoter-specific receptor function. We have identified critical amino acid residues within GR which, when mutated, genetically distinguish the action of GR at these promoters. Taken together, the data indicate that the presence of only the receptor and the ligand is not sufficient to allow activation of transcription. An additional system of regulation influences receptor action in both a tissue- and promoter-selective fashion, suggesting that multiple, regulated surfaces of the receptor respond to the cellular environment and determine the spectrum of GR activities. These functional surfaces may be induced or regulated by ligand binding, by the DNA sequence to which receptor is bound, or by the nonreceptor factors resident at the promoter or in the tissue.

Long-term culture of primary B cells and in vitro expression of an exogenous gene.

Efficient introduction and expression of exogenous genes into primary B cells is very important to study B-cell biology and is essential for gene therapy. These efforts have often been impeded by the lack of availability of a simple culture condition for growth and proliferation of primary B cells as well as the lack of vehicles for efficient introduction of genes of interest. In this communication, we have developed a culture condition that supports the growth of primary B cells from beta-gal-immunized mouse spleen for 30 days without the aid of feeder layers. During this period, B cells secreted polyclonal antibodies into the medium. To study expression of an exogenous reporter gene, human growth hormone (hGH) was introduced into cultured B cells using retroviral vectors. hGH was expressed up to 21 days in the absence of drug selection and the infected cells continued to secrete immunoglobulins into the medium.

The major 45-kDa protein of the yeast mitochondrial outer membrane is not essential for cell growth or mitochondrial function.

As part of an analysis of the function and assembly of the mitochondrial outer membrane, we have cloned and characterized the yeast gene encoding a 45-kDa polypeptide (OM45) which is a major constituent of this membrane. The nuclear gene was isolated by immunological screening of plaques of recombinant phage lambda gt11 containing fragments of yeast genomic DNA using an antibody against OM45. Determination of the nucleotide sequence of the DNA fragment isolated by this approach revealed a single open reading frame of 1179 base pairs which encodes a protein having a predicted molecular mass of 44.6-kDa. Disruption of the OM45 gene in haploid yeast cells eliminated the expression of OM45. The mutant strain showed no apparent defect in cell viability, growth, mitochondrial function, or mitochondrial protein import.